Transformation of <Emphasis Type="Italic">Campanula</Emphasis> by wild type <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>

Compact growth is an important quality criterion in horticulture. Many Campanula species and cultivars exhibit elongated growth which is suppressed by chemical retardation and cultural practice during production to accommodate to the consumer’s desire. The production of compact plants via transformation with wild type Agrobacterium rhizogenes is an approach with great potential to produce plants that are non-GMO. Efficient transformation and regeneration procedures vary widely among both plant genera and species. Here we present a transformation protocol for Campanula. Hairy roots were produced on 26–90% of the petioles that were used for transformation of C. portenschlagiana (Cp), a C. takesimana × C. punctata hybrid (Chybr) and C. glomerata (Cg). Isolated hairy roots grew autonomously and vigorously without added hormones. The Cg hairy roots produced chlorophyll and generated plantlets in response to treatments with cytokinin (42 µM 2iP) and auxin (0.67 µM NAA). In contrast, regeneration attempts of transformed Cp and Chybr roots lead neither to the production of chlorophyll nor to the regeneration of shoots. Agropine A. rhizogenes strains integrate split T-DNA in T_L- and T_R-DNA fragments into the plant genome. In this study, regenerated plants of Cg did not contain T_R-DNA, indicating that a selective pressure against this T-DNA fragment may exist in Campanula.     

Asparagine synthetase genes (<Emphasis Type="Italic">AsnS1</Emphasis> and <Emphasis Type="Italic">AsnS2</Emphasis>) in durum wheat: structural analysis and expression under nitrogen stress

Wheat is one of the most widely grown cereal crops based on the amount of calories it provides in the human diet. Durum wheat (Triticum turgidum ssp. durum) is largely used for production of pasta and other products. In order to use genetic knowledge to improve the understanding of N-use efficiency, we carried out, for the first time in durum wheat, the isolation and the characterization of four members of the asparagine synthetase (AsnS) gene family. Phylogenetic inference clustered the Ttu-AsnS1 (1.1 and 1.2) and Ttu-AsnS2 (2.1 and 2.2) genes in AsnS gene class I, which is present in monocots and dicots. Class I genes underwent a subsequent duplication leading to the formation of two subgroups. Plants of Svevo cultivar were grown under N-stress conditions and expression of the four AsnS genes was investigated at three developmental stages (seedling, booting, and late milk development), crucial for N absorption, assimilation and remobilization. AsnS1 genes were down-regulated in N-stressed roots, stems and leaves during seedling growth and booting, but seemed to play a role in N remobilization in flag leaves during grain filling. AsnS2 genes were scarcely expressed in roots, stems, and leaves. In N-stressed spikes there was no differential expression in any of the genes. The genes were mapped in silico using a durum wheat SNP map, assigning Ttu-AsnS1 genes to chromosome 5 and Ttu-AsnS2 to chromosome 3. These findings provide a better understanding of the role of ASN genes in response to N stress in durum wheat.     

Genetic transformation of pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> L.) and screening transgenic progenies based on lateral root inhibition

Production of transgenic pigeonpea is becoming increasingly important, but the methods currently employed in production and subsequent screening still requires improvement. Here, we describe Agrobacterium-mediated genetic transformation of pigeonpea with reporter uidA (gus) gene and selectable marker, neomycin phospho-transferase (nptII) gene. Histochemical assay demonstrate localization of gus activity in cells and transformed plants. Overall, a transformation frequency of 0.33% was achieved using the protocol. Grafting of in vitro-regenerated healthy shoots indicates higher survival percent (72.6%), when stock and scion are of the same variety. Seeds harvested from primary transgenic plants can be screened based on lateral root inhibition strategy. Approximately 87% of the screened T₁ plants were found to be PCR positive. In conclusion, in vitro grafting of transgenic pigeonpea shoots leads to better plant establishment and screening based on lateral root inhibition leads to quick identification of positive segregants.     

Development and application of a molecular marker for TMV resistance based on <Emphasis Type="Italic">N</Emphasis> gene in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

Tobacco mosaic virus (TMV) caused serious loss in yield and quality of tobacco every year. It is a long-term goal to improve the tobacco resistance against TMV by tobacco breeding. N gene was the firstly reported TMV-resistant gene, which showed resistance against all Tobamoviruses except the Ob stain and belonged to the toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat class of plant resistance (R) genes. At present, N gene had already been widely used in tobacco conventional breeding, but there is rare available molecular maker used in marker-assisted selection of TMV resistance. In this study, we designed a pair of primers that specific amplify N gene fragment based on the sequence of N gene intron III, named N-marker. Then, we identified TMV resistance by two selecting methods, PCR with N-marker and inoculated with the TMV-C strain. Results from the two method showed that (1) 13 varieties among 67 tobacco varieties displayed hypersensitive reaction when inoculated with the TMV-C strain, also contained N gene fragments screened by PCR with N-marker; (2) 105 strains of 200 BC1 strains showed resistance against TMV when inoculated with TMV-C strain, meanwhile, 103 of the 105 strains contained N gene fragment verified by PCR with N-marker. Therefore, the N-marker is reliable for high throughput screening of germplasm resources and tobacco breeding materials in selection of N-mediated TMV resistance. Our study not only developed a molecular marker for tobacco breeding, but also identified new germplasm resources that are resistant to TMV.     

Linkage between genes for hairy first leaf and chlorophyll deficiency in Brassica oleracea

The fourth linkage group of B. oleracea L. has two genes: Hr-1, (hairy first leaf), a dominant seedling marker from “Dwarf Green” curly kale, and pg-2, (pale green seedling), a recessive chlorophyll mutant from green sprouting broccoli. Recombination between Hr-1 and pg-2 ranged from 7.4 to 20.1% in the six progenies studied, with a mean of 13.15±0.68%. Hr-1 segregated independently of the three other linkage groups (two genes of each were tested) and of two unlocated genes for male sterility.     

Biochemical side effects of the herbicide FINALE<Superscript>®</Superscript> on <Emphasis Type="Italic">bar</Emphasis> gene-containing transgenic pineapple plantlets

Pineapple is one of the most important tropical fruits and therefore intensive genetic improvement programs are being carried out in many countries, including Cuba. Our research team has previously introduced the bar gene, along with chitinase and AP24 genes, into the pineapple genome. Herein, we report on the biochemical side effects of the herbicide FINALE^® on these transgenic plantlets during hardening. Levels of aldehydes and chlorophylls, and peroxidase activity were recorded. The transformed clone studied here, not sprayed with FINALE^®, showed the following side effects because of transgenesis only. Levels of malondialdehyde, other aldehydes, chlorophyll b, and total chlorophyll pigments decreased. The most remarkable biochemical differences between transgenic and non-transgenic plantlets after application of FINALE^® follow. Levels of malondialdehyde and other aldehydes in transgenic material were not decreased by FINALE^®, perhaps because these levels were already low as a result of transformation. FINALE^® increased peroxidase activity in transgenic plantlets but such increase was higher in non-transgenic material. The herbicide increased contents of chlorophyll pigments (a, b, total) in transformed plantlets. However, as expected, non-transgenic plantlets decreased levels of chlorophylls (a, b, total) after application of FINALE^®. The genetic transformation of pineapple with the bar gene not only conferred resistance to the herbicide FINALE^®, but also promoted other biochemical changes.     

Resistance to <Emphasis Type="Italic">Cucumber green mottle mosaic virus</Emphasis> in <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Cucumber green mottle mosaic virus (CGMMV) is a severe threat for cucumber production worldwide. At present, there are no cultivars available in the market which show an effective resistance or tolerance to CGMMV infection, only wild Cucumis species were reported as resistant. Germplasm accessions of Cucumis sativus, as well as C. anguria and C. metuliferus, were mechanically infected with the European and Asian strains of CGMMV and screened for resistance, by scoring symptom severity, and conventional RT-PCR. The viral loads of both CGMMV strains were determined in a selected number of genotypes using quantitative RT-PCR. Severe symptoms were found following inoculation in C. metuliferus and in 44 C. sativus accessions, including C. sativus var. hardwickii. Ten C. sativus accessions, including C. sativus var. sikkimensis, showed intermediate symptoms and only 2 C. sativus accessions showed mild symptoms. C. anguria was resistant to both strains of CGMMV because no symptoms were expressed and the virus was not detected in systemic leaves. High amounts of virus were found in plants showing severe symptoms, whereas low viral amounts found in those with mild symptoms. In addition, the viral amounts detected in plants which showed intermediate symptoms at 23 and 33 dpi, were significantly higher in plants inoculated with the Asian CGMMV strain than those with the European strain. This difference was statistically significant. Also, the amounts of virus detected over time in plants did not change significantly. Finally, the two newly identified partially resistant C. sativus accessions may well be candidates for breeding programs and reduce the losses produced by CGMMV with resistant commercial cultivars.     

Induced expression of selected plant defence related genes in pot azalea, <Emphasis Type="Italic">Rhododendron simsii</Emphasis> hybrid

A set of putative marker genes to study plant defense responses against Polyphagotarsonemus latus, a key pest in the production of Rhododendron simsii hybrids, was selected and validated. Genes belonged to the biosynthetic pathway of phytohormones jasmonic acid (JA) (RsLOX, RsAOS, RsAOC, RsOPR3 and RsJMT) and salicylic acid (SA) (RsPAL and RsICS). Furthermore, RsPPO, a putative marker gene for oxidative stress response was successfully cloned from R. simsii. A CTAB-based extraction protocol was optimized to assure excellent RNA quality for subsequent RT-qPCR analysis. The RT-qPCR protocol was extensively tested and RsRG7 and RsRG14 were selected as reference genes from a geNorm pilot study. Validation of the marker genes was done after application with elicitors [methyl jasmonate (MeJA), coronatine, β-aminobutyric acid and acibenzolar-Smethyl] or wounding. Both 100 μM MeJA and 0.1 μM coronatine had a significant effect on the expression of all marker genes. Foliar application of MeJA on the shoots resulted in a significantly earlier response when compared to root application and subsequent sampling of the shoots. Expression patterns after MeJA treatment were generally the same in six R. simsii genotypes: ‘Nordlicht’, ‘Elien’, ‘Aiko Pink’, ‘Michelle Marie’, ‘Mevrouw Gerard Kint’ and ‘Sachsenstern’. Wounding resulted in the same expression patterns as MeJA treatment except for RsJMT. None of the genotypes showed a significant induction of the latter gene 6 h upon wounding. Findings of these experiments indicated that the tolerant genotype ‘Elien’ has low basal expression levels of RsPPO. This might be the first step towards the breeding of mite-tolerant genotypes.     

Resistance screening of breeding lines and commercial tomato cultivars for <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> and <Emphasis Type="Italic">M. javanica</Emphasis> populations (Nematoda) from Ethiopia

Soil and root samples were collected from major tomato growing areas of Ethiopia during the 2012/2013 growing season to identify root-knot nematode problems. DNA-based and isozyme techniques revealed that Meloidogyne incognita and M. javanica were the predominant Meloidogyne species across the sampled areas. The aggressiveness of different populations of these species was assessed on tomato cultivars Marmande and Moneymaker. The two most aggressive populations of each species were selected and further tested on 33 tomato genotypes. The resistance screening and mechanism of resistance was performed after inoculation with 100 freshly hatched (<24 h) second-stage juveniles (J2). Eight weeks after inoculation the number of egg masses produced on each cultivar was assessed. For the resistance mechanism study, J2 penetration and their subsequent development inside the tomato roots were examined at 1, 2, 4 and 6 weeks after inoculation. On both cultivars Marmande and Moneymaker all M. incognita and M. javanica populations formed a high number of egg masses indicating highly aggressive behaviour. Populations from ‘Jittu’ and ‘Babile’ for M. incognita and ‘Jittu’ and ‘Koka’ for M. javanica were selected as most aggressive. None of the 33 tomato genotypes were immune for these M. incognita and M. javanica populations. However, several tomato genotypes were found to have a significant effect on the number of egg masses produced indicating possible resistance. For M. javanica populations there were more plants from cultivars or breeding lines on which no egg masses were found compared to M. incognita populations. The lowest number of egg masses for both populations of M. incognita was produced on cultivars Bridget40, Galilea, and Irma while for M. javanica it was on Assila, Eden, Galilea, Tisey, CLN-2366A, CLN-2366B and CLN-2366C. Tomato genotypes, time (weeks after inoculation) and their interaction were significant sources of variation for J2 penetration and their subsequent development inside the tomato roots. Differential penetration was found in breeding lines such as CLN-2366A, CLN-2366B and CLN-2366C, but many of the selected tomato genotypes resistance for the tested M. incognita and M. javanica populations were expressed by delayed nematode development. Therefore, developing a simple screening technique to be used by local farmers or extension workers is crucial to facilitate selection of a suitable cultivar.     

Tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation studies in four commercially important indica rice cultivars

This research was undertaken to find an efficient tissue culture system and Agrobacterium-mediated genetic transformation method for recalcitrant indica rice cultivars. For this, mature seeds of commercially important indica rice varieties, ASD16, ADT43, IR 64, and Pusa Basmati were cultured on MS and N6 medium supplemented with 2 mg l^-1 2, 4-D + 30 g l^-1 sucrose. The calli grown in N6 medium showed better friability and embryogenic response. Out of the four varieties tested, ASD16 and IR64 showed better callusing and embryogenic capacity as compared to ADT43 and Pusa Basmati. For genetic transformation studies, embryogenic calli of all the cultivars were co-cultivated with the Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCambia 1305.1 with GUS gene. GUS assay was performed for the putative transformed calli and its activity was found to be qualitatively higher in ASD16 and IR64 than the other two varieties. The best responsive ASD16 transformed calli was regenerated and the putative transgenic lines were regenerated. ASD16 transformed calli were confirmed by GUS assay. PCR analysis confirmed the presence of both GUS and HPT genes in ASD16 transgenic lines.     

Complementary DNA (cDNA) cloning and functional verification of resistance to head smut disease (<Emphasis Type="Italic">Sphacelotheca reiliana</Emphasis>) of an NBS–LRR gene <Emphasis Type="Italic">ZmNL</Emphasis> in maize (<Emphasis Type="Italic">Zea mays</Emphasis>)

The nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family comprises the largest number of known disease resistance (R) genes and is one of the largest gene families in plants. In the present study, the full-length cDNA of ZmNL (GenBank Accession Number KF765443) was isolated using Rapid Amplification of cDNA Ends. The nucleotide sequence of ZmNL contains an open reading frame of 3156 bp that encodes the ZmNL protein, which is comprised of 1051 amino acid residues. This putative protein has high homology to other known resistance proteins (84% to Triticum aestivum LR10) and belongs to the CC–NBS–LRR type R gene family. The ZmNL gene was introduced into the maize inbred line of Huangzao4 which was highly susceptible to head smut under the control of the maize ubiquitin promoter by Agrobacterium-mediated transformation. The head smut disease incidence of 3 T₂ transgenic lines was significantly reduced (by 18.38–29.40%) compared with the wild type, which indicated that the overexpression of ZmNL gene in maize enhanced the resistance to the fungus Sporisorium reilianum (Kühn) Clint of these plants.     

Breeding of Lignocellulosic Bioethanol Feedstock

As sustainability becomes a pivotal issue worldwide, biofuel from plants has been highlighted as an alternative to energy from fossil fuels. In the current review, we focused on improving the efficiency of lignocellulosic bioethanol production from high dry matter-producing Miscanthus and switchgrass species by understanding these species’ genetic traits and responses to various stresses. The most recent findings regarding biomass quality and bioethanol conversion processes are discussed in this review, including goals of current feedstock breeding programs, followed by up-to-date genetics and genomics resources to provide optimal breeding approaches for Miscanthus and switchgrass species. We revisited previous breeding approaches using bmr mutations, ethyl methanesulfonate (EMS), next generation sequencing (NGS), genome-wide association study (GWAS), and transgenic resources, which can be a basis for improving sustainable biomass and biofuel production through these two species. This review may provide background for researchers and breeders to further improve breeding approaches.     

Broad spectrum insect resistance and metabolites in close relatives of the cultivated tomato

Wild relatives of tomato possess effective means to deal with several pests, among which are a variety of insects. Here we studied the presence of resistance components against Trialeurodes vaporariorum, Myzus persicae, Frankliniella occidentalis, and Spodoptera exigua in the Lycopersicon group of Solanum section Lycopersicon by means of bioassays and comprehensive metabolite profiling. Broad spectrum resistance was found in Solanum galapagense and a few accessions of S. pimpinellifolium. Resistance to the sap sucking insects may be based on the same mechanism, but different from the caterpillar resistance. Large and highly significant differences in the leaf metabolomes were found between S. galapagense, containing type IV trichomes, and its closest relative S. cheesmaniae, which lacks type IV trichomes. The most evident differences were the relatively high levels of different methylated forms of the flavonoid myricetin and many acyl sucrose structures in S. galapagense. Possible candidate genes regulating the production of these compounds were identified in the Wf-1 QTL region of S. galapagense, which was previously shown to confer resistance to the whitefly B. tabaci. The broad spectrum insect resistance identified in S. galapagense will be very useful to increase resistance in cultivated tomato.     

Quantitative trait loci affecting intensity of violet flower colour in potato

Anthocyanins occur in potato tuber skin and flesh, sprouts, leaves, stems and flowers. The goal of this study was to identify genomic regions and candidate gene alleles key for accumulation of anthocyanins in potato corolla in various quantities. QTL analyses were performed in two mapping populations segregating for flower colour intensity and candidate genes were identified on the basis of function and location (chalcone isomerase, chi; chalcone synthase, chs) or location (RNA-dependent RNA polymerase 1, RDR1). We detected three and four QTL affecting the violet flower colour intensity using the two mapping populations, respectively. In both populations a locus F, necessary for violet flower colour, segregated and we used different approaches to differentiate the qualitative effect of this locus and to detect the genetic factors affecting the quantitative flower colour intensity. The strongest QTL and the only one common for the two mapping populations was located on chromosome V. The role of all three candidate genes, chi, chs and RDR1, in control of flower colour intensity is supported to different extents by the performed genetic analyses. The most important QTL on chromosome V is most likely in the same position as the QTL for anthocyanin tuber flesh coloration described previously, which indicates that the natural variation in some biosynthetic and/or regulatory genes may influence anthocyanin levels in multiple tissues.     

Creating cold resistant strawberry via interploidy hybridization between octoploid and dodecaploid

Strawberry cultivars showed limited cold resistance in the Northeast of China, while we obtained a synthetic dodecaploid strawberry hybrid ‘YH15-10’ (2n = 12x = 84) which showed sufficient cold resistance in this area. The reciprocal crosses between F. × ananassa cv. ‘Allstar’ (2n = 8x = 56) and ‘YH15-10’ (2n = 12x = 84) were carried out to select cold resistant strawberry in this study. The 134 seedlings were obtained from the cross of Allstar × YH15-10, while failed in its reciprocal cross. The 30 randomly selected seedlings were examined in terms of morphological characters, chromosome numbers and cold resistance. Most morphological characters were widely separated among F1 progeny with a high broad-sense heritability, which showed that these variations mainly resulted from genetic effect. Some hybrids exhibited heterosis, especially in growth vigor and runner production. Among the 30 tested hybrids, 28 decaploids (2n = 10x = 70), one octoploid (2n = 8x = 56) and one enneaploid (2n = 9x = 63) were observed. The 63.3% hybrids demonstrated higher cold resistance than that of ‘Allstar’ at P < 0.05. These high polyploidy strawberries have potential values in commercial production and modern cultivar improvement.     

<Emphasis Type="Italic">In vivo</Emphasis> Acclimatization Responses of <Emphasis Type="Italic">Platycodon grandiflorum</Emphasis> For. Duplex to Different Soil Types and Environmental Factors

This study aimed to evaluate the effects of soil types and environmental factors for optimum conditions of seedlings growth of the Platycodon grandiflorum for establishing the in vivo acclimatization system of regenerated plants derived from the in vitro culture. P. grandflorum seedlings were transferred to the in vivo condition and acclimatized under different soil types, light intensities, and various temperatures. Changes caused by environmental factors and soil types in plant growth viz. plant height, leaf width, leaf length, stem diameter, number of leaves, branches and nodes were recorded in this study. Among the nine types of soil, the best growth performances were obtained from the soil type SVP (Soil mixed with horticultural bed soil, vermiculite, and perlite @ 2:1:1). Seedlings of P. grandiflorum showed the best growth at higher levels of light intensity (60 μmol·m^-2·s^-1). In contrast, P. grandiflorum seedlings showed the best growth response at a moderate level of temperature (25°C). Collectively, the present study provides a better understanding of the responses of growth characteristics in P. grandiflorum seedlings exposed to various soil types, light intensities, and temperatures.     

Mapping QTLs for plant height and flowering time in a Chinese summer planting soybean RIL population

Soybean is a primary source of plant oil and protein and has a high nutritional value. Plant height (PH) and flowering time (FT) are two important agronomic traits in breeding programs for soybean. In this study, we mapped QTLs associated with PH and FT in three environments using a population with determinate growth including 236 recombinant inbred lines (NJZY-RIL) derived from a cross between two summer planting varieties, ZXD and NN1138-2. A high-density genetic map with 3255 SLAF-markers was constructed that spanned 2144.85 cM of the soybean genome with an average marker distance of 0.66 cM. Altogether, six QTLs controlling PH and eleven QTLs controlling FT were mapped using mixed-model-based composite interval mapping and composite interval mapping methods. qPH-1-1 and qFT-15-2 were two novel main effect QTLs identified in this study; qFT-6-2, qFT-15-2, qFT-16-1, qPH-1-1, qPH-15-1 and qPH-16-1 were consistently detected across environments and by the two mapping methods. Two pairs of QTLs, qFT-15-2 and qPH-15-1 as well as qFT-16-1 and qPH-16-1, which were located in the same marker interval on chromosomes 15 and 16, respectively, were found to have close linkage or pleiotropy. These results may increase our understanding of the genetic control of PH and FT in soybean and provide support for implementing marker-assisted selection in developing soybean cultivars with high yield and early maturity in summer planting regions.     

Fine mapping the <Emphasis Type="Italic">BjPl1</Emphasis> gene for purple leaf color in B2 of <Emphasis Type="Italic">Brassica juncea</Emphasis> L. through comparative mapping and whole-genome re-sequencing

Purple plants with higher anthocyanin content have attracted increasing attention in recent years due to their advantageous biological functions and nutritional value. A spontaneous mutant with purple leaves, designated 1280-1, was discovered in Brassica juncea line 1280. A previous genetic analysis indicated that the purple leaf trait in 1280-1 was controlled by a dominant gene (BjPl1). In the present study, an analysis of total anthocyanin content further indicated that the purple leaf trait was controlled by a complete dominance gene. According to a survey of 426 primers available from public resources, BjPl1 was assigned to linkage group B2 of B. juncea. In the early stage of this research, based on comparative mapping in Brassica, two simple sequence repeat (SSR) markers developed from A2 of B. rapa delimited the BjPl1 gene to a 0.7-cM genetic interval in the corresponding linkage map. According to information on the B. juncea genome released recently, the location of BjPl1 was further narrowed to a 225-kb interval (17.74–17.97 Mb). Within the target region, whole-genome re-sequencing identified two candidate regions (17.74–17.78 Mb and 17.93–17.96 Mb). Through Blast analysis of the two candidate intervals, four homologous anthocyanin biosynthetic genes were identified and localized to a 17.93–17.96 Mb interval of B2 (approximately 27 kb), which might include BjPl1. This work lays the foundation for the isolation of BjPl1 and will further improve our understanding of the molecular mechanisms of the anthocyanin metabolic pathway in Brassica.     

Genotyping-by-sequencing of waxy and glossy near-isogenic broccoli lines

Wild-type Brassica oleracea L. have matte blue-green leaves caused by an interaction between leaf pigmentation and a waxy bloom coating the surface. Glossy mutants have reduced and/or altered epicuticular wax giving the leaves a shiny green appearance and have been identified in most B. oleracea crop varieties, including cauliflower, kale, broccoli, Brussels sprouts, cabbage, and collard. The genetic basis of glossy mutants has not been studied in B. oleracea. Glossiness can confer resistance to multiple herbivores mediated by modification of herbivore feeding behavior and foraging efficiency of their predators. The USDA-ARS-U.S. Vegetable Laboratory released two pairs of near-isogenic broccoli lines (NILs) that visibly differ only for glossiness, providing ideal germplasm for the genetic study of epicuticular wax in B. oleracea. Genotyping-by-sequencing resulted in hundreds of polymorphisms between each pair of NILs. Polymorphisms were identified in or near three different wax synthesis genes suggesting the two glossy mutants were caused by alteration of different steps of the pathway.